1. Technical Field of the Invention
The present invention involves the creation of defined chromosomal deficiencies, inversions and duplications using Cre recombinase in embryonic stem cells and transmitted into the mouse germ line. In the present invention, these chromosomal reconstructions can extend up to 3-4 cM. Chromosomal rearrangements are the major cause of inherited human disease and fetal loss. Further, chromosomal translocations and deletions are recognized as major genetic changes that are causally involved in neoplasia. Chromosomal variants such as deletions and inversions are exploited commonly as genetic tools in diploid it organisms such as Drosophila. In diploid organisms, such deficiencies are exploited in genetic screens because a small portion of the genome is functionally hemizygous. Thus, a mutation which would normally be recessive and masked by the wildtype allele in a diploid context will be dominant and detectable in the haploid state. In the mouse, deficiencies have not, up to now, been available generally; thus, screens for recessive mutations are nonexistent or particularly cumbersome. However, the present invention provides methods to engineer mice and cell lines with defined regions of segmental haploidy. Such mice are useful for genetic screening and provide accurate models of human chromosomal diseases.
2. The Prior Art
Inherited chromosomal rearrangements such as inversions, duplications and deficiencies are responsible for a significant fraction of human congenital disease. Chromosomal changes also occur somatically and are associated with neoplastic disease. Defining the causal genetic alteration in a region of the genome associated with chromosomal rearrangements can be relatively straightforward if the affected gene lies in the breakpoint of an inversion or translocation. However, in cases of duplications and deficiencies, the specific genetic lesion(s) associated with pathological chromosomal changes are much harder to identify. Still, the generation of animal models that accurately recapitulate the genetic lesion would facilitate the study of disease and could be very helpful in the efforts to dissect specific gene-function relationships in multigene syndromes.
In diploid organisms such as Drosophila, chromosomal deficiencies are commonly exploited in genetic screens because a small portion of the genome is functionally hemizygous. Thus, a mutation which would be recessive and masked by the wildtype allele in the diploid context will be dominant and therefore readily detectable in the haploid state. In the mouse, deficiencies are not available generally. Despite the limited number of deficiencies available in the mouse, the potential for the detailed analysis of a genetic interval using these deficiencies has been demonstrated clearly. See Holdener-Kenny, et al., BioEssays, 14:831-39 (1992), which is hereby incorporated by reference.
Deficiencies that are available currently in the mouse genome were generated at random using ionizing irradiation. Although conventional gene targeting technology in embryonic stem (ES) cells can generate virtually any type of mutation, including deletions of up to 20 kb, it has not been possible to delete substantially larger fragments by using standard methodology. Likewise, the technology required to construct large inversions and duplications has not been established.
One mechanism by which chromosomes may be engineered is by the use of Cre recombinase. Cre recombinase has been used in mammalian cell lines and in vivo to delete or invert sequences between the 34 base pair recognition sequences, loxP sites, placed a few kb apart on the same chromosome. The recombination is initiated by Cre proteins which bind to 13-bp inverted regions in the loxP sites and promote synapses or joining of a pair of sites. Next, the Cre proteins catalyze strand exchange between the pair of sites within an asymmetric 8-bp central spacer sequence by concerted cleavage and rejoining reactions, involving a transient DNA-protein covalent linkage. Smith, et al., Nature Genetics, 9:376-385 (1995); Gu, et al., Science, 265:103-06 (1994) and Sauer, Nucl. Acids Res., 17:147-61 (1989) (both of these references are hereby incorporated by reference). Additionally, recombinases have been used to induce mitotic recombination between homologous and non-homologous chromosomes in Drosophila, plants and mammalian cells. Embryonic stem cell technology has become a powerful tool for defining the function of mammalian genes, but mainly has been restricted to the mutation of single genes. Replacement vectors have been used to construct deletions of up to 19 kb; however, utilizing the same strategy to construct larger deletions ( greater than 60 kb) has not been successful. In the present invention, the generation and direct selection of deletions, duplications and inversions, ranging from 90 kb to 3-4 cM, in ES cells is demonstrated.
The method of the present invention is based on consecutive gene targeting of two recombination substrates to the deletion endpoints and the subsequent induction of recombination mediated by the Cre recombinase. This method generates a positive selectable marker allowing for the direct selection of clones with the desired chromosome structures. Despite the multitude of steps involved in generating these rearrangements in ES cells, deletion and duplication alleles have been transmitted into the mouse genome.
One object of the present invention is a method for causing a large-scale chromosomal rearrangement by first deleting a portion of genetic material.
An additional object of the present invention is a targeting vector system capable of inserting into two endpoint regions constraining a desired chromosomal deletion.
Thus in accomplishing the foregoing objects, there is provided in accordance with one aspect of the present invention a method for deleting a selected region of genetic material in cells comprising the steps of: inserting a first selection cassette at a 5xe2x80x2 end of said selected region using conventional gene targeting methods, said first selection cassette comprising a first selectable marker, a first loxP recombination site, and a first portion of a second selectable marker; selecting cells expressing said first selectable marker; inserting a second selection cassette at a 3xe2x80x2 end of said selected region using conventional gene targeting methods, said second selection cassette comprising a third selectable marker, a second loxP recombination site, and a remaining portion of said second selectable marker; selecting cells expressing said third selectable marker; expressing Cre recombinase to produce recombination between said first and second loxP sites; and selecting cells expressing said second selectable marker.
Specific embodiments of the above method can include a puromycin resistance gene as the first selectable marker, a functional Hprt gene as the second selectable marker, and a neomycin resistance gene as the third selectable marker. Numerous other selectable markers will work, their presence in the particular deletion strategy is merely to aid cell selection. In other preferred embodiments, the first selectable marker is a puromycin resistance gene. In still other preferred embodiments, the second selectable marker is a functional Hprt gene. And in still other preferred embodiments, the third selectable marker is a neomycin resistance gene.
In still other preferred embodiments, the cells referred to above are embryonic stem cells, though significant, they need not be stem cells. In other preferred embodiments, the cells are embryonic stem cells, and said cells develop into mice. And in yet other preferred embodiments, the cells are embryonic stem cells, and said cells are maintained as cell lines.
In yet another preferred embodiment, a viral vector is used to replace either or both native sequences of DNA. In one embodiment, this virus is a retrovirus. In yet another embodiment, the viral vector referred to above has a provirus structure comprising a cassette in turn comprising: an hprtxcex945xe2x80x2 cassette, a loxP site, and a puromycin resistance gene.
In yet another particularly preferred embodiment, the method for deleting a portion of chromosomal material in cells wherein the targeting vectors are a first targeting vector for replacing said first native sequence of DNA at said 5xe2x80x2 end, comprising: a genomic insert cloned into the vector of about 7.5 kb; a tyrosinase minigene; a Neor gene; a 5xe2x80x2 hprt fragment; and a loxP site embedded into said hprt fragment; and a second targeting vector for replacing said second native sequence of DNA at said 3xe2x80x2 end, comprising: a genomic insert cloned into the vector of about 8.5 kb; a K14-Agouti gene; a Puror gene; a 3xe2x80x2 hprt fragment; and a loxP site embedded into said hprt fragment.
In one particularly preferred embodiment of the second aspect of the present invention, there is provided a replacement vector system comprising a first targeting vector for replacing said first native sequence of DNA at said 5xe2x80x2 end, comprising a genomic insert cloned into the vector of about 7.5 kb; a tyrosinase minigene; a Neor gene; a 5xe2x80x2 hprt fragment; and a loxP site embedded into said hprt fragment; and a second targeting vector for replacing said second native sequence of DNA at said 3xe2x80x2 end, comprising: a genomic insert cloned into the vector of about 8.5 kb; a K14-Agouti gene; a Puror gene; a 3xe2x80x2 hprt fragment; and a loxP site embedded into said hprt fragment.